1. Field of the Invention
The present invention relates to a liquid composition and a method of introducing heme, and/or hemoproteins, and/or heme-hemoprotein complexes into the body. More particularly, incorporating heme, hemoprotein or a heme-hemoprotein complex into a liquid coacervate system, provides a composition that unexpectedly overcomes the intrinsic instability of heme and therefore is useful in augmenting the oxygen transport capacity of the body; serves as an agent in the treatment of several anemias; and acts as an oxygen-carrying plasma volume expander.
2. Brief Description of the Prior Art
Heme is known to be an unstable molecule However, the incorporation of heme and/or hemoprotein into the coacervate system of the present invention not only unexpectedly improves the stability of heme but also surprisingly provides a composition that is useful in treating blood loss, iron deficiency anemias and for providing oxygen to the body.
The molecular structure of heme, the role of heme in the production of hemoglobin and the physiology of heme are well known. The use of heme and the heme-hemoprotein complex in accordance with the present invention is not taught by the prior art of known blood substitutes using hemoglobin. In the hemoglobin molecule, the protein globulin provides the heme certain physical-chemical properties that increases the stability of heme. Globulin also facilitates oxygen binding and release, and is involved in antigen-antibody reactions.
In the present invention, rather than globulin, the liquid coacervate system, and particularly the surfactants contained therein, is used to stabilize the heme and/or hemoprotein. In addition, the liquid coacervate system composition of the present invention avoids the antigen-antibody reactions associated with hemoglobin, thereby reducing or eliminating the possibility of toxic reactions. The prior art does not teach a composition comprising either heme, a hemoprotein or a complex consisting of heme and a hemoprotein in a two-phase liquid coacervate system.
Blood substitutes based upon a two-phase liquid coacervate system and stroma-free hemoglobin have been prepared previously. However, the present invention, also based upon a coacervate system, utilizes heme, and/or hemoprotein and/or a heme-hemoprotein complex instead of the stroma-free hemoglobin used in the prior art. In accordance with an important feature of the present invention, the molecular structure of heme is identical in all mammals, thereby assuring a virtually unlimited, inexpensive supply of heme. In contrast, the supply of human hemoglobin is limited and expensive and is acceptable for human use only after extensive and costly processing. Therefore, the use of heme, and/or hemoprotein and/or a heme-hemoprotein complex produces non-toxic, effective, and economical compositions compared to presently used products containing stroma-free hemoglobin.
The disclosures of the following Ecanow and Ecanow et al. patents and patent applications are hereby incorporated into this specification by reference.
The Ecanow U.S. Pat. No. 4,343,797 discloses an intravenous synthetic blood substitute comprising a two-phase heterogeneous coacervate system and stroma-free hemoglobin.
The Ecanow et al. U.S. Pat. No. 4,439,424 discloses intravenous synthetic whole blood products in the form of coacervate systems, containing a coacervate phase, an equilibrium bulk water phase and stroma-free hemoglobin.
The Ecanow et al. U.S. patent application Ser. No. 512,917, filed July 12, 1983, now U.S. Pat. No. 4,547,490 discloses a composition wherein a coacervate system is produced from lecithin dispersed in an aqueous solution containing sodium chloride and albumin, then stroma-free hemoglobin and a non-polar or semi-polar solvent, such as n-butyl alcohol, is added to the coacervate system.
The Ecanow et al. U.S. patent application Ser. No. 811,675, filed Dec. 20, 1985now U.S. Pat. No. 4,738,952 discloses a synthetic whole blood wherein the coacervate system is produced from lecithin dispersed in an aqueous solution containing albumin and sodium chloride. The coacervate system disclosed provides for more effective use of the pyridoxylated-polymerized hemoglobin component of the composition.
The Ecanow U.S. Pat. No. 4,539,204 describes a synthetic blood substitute based on stroma-free hemoglobin and on a coacervate system comprised of two gelatins having different isoelectric points.
The Ecanow U.S. Pat. No. 4,596,788 discloses a synthetic whole blood based upon a coacervate system including gelatin, lecithin, and stroma-free hemoglobin.
The Ecanow U.S. patent application Ser. No. 835,550, filed Mar. 3, 1986,discloses an oral dosage form of insulin based on the use of a coacervate system comprised of lecithin, albumin, and insulin.
The Ecanow U.S. patent application Ser. No. 711,066,now abandoned, filed Mar. 12, 1985, discloses an oral dosage form of atrial peptides, and peptides in general, based on a coacervate including lecithin, albumin, and atrial peptides.
The prior art further includes a publication by Hasegawa describing a blood substitute comprising an amphiphilic heme embedded in a liposome preparation: Hasegawa, E., et al. "Synthesis of Polymeric Liposome Embedded Heme (PLH) and its Possibility as New Synthetic Oxygen Carrier", J. Pharmacobiodynamics, 9, No. 9, 117, 1986. However, the composition described by Hasegawa differs from the composition of the present invention in several significant respects. Hasegawa uses a liposome to encapsulate the heme molecule, in contrast to the present invention teaching the use of a coacervate system to incorporate the heme, and/or hemoprotein and/or a heme-hemoprotein complex. This distinction is substantial because in the Hasegawa composition, the heme component is enclosed by lecithin while in the present invention, heme is bound to a surface active agent, such a albumin, as it is in natural blood. In addition, the half-life of heme in liposome systems is significantly shorter than the half-life of heme in coacervate systems.
The liposome vehicle used by Hasegawa and the coacervate system of the present invention are fundamentally different in that the two compositions have different components, possess different properties, and yield different end products.
Generally, the differences between liposomes of Hasegawa and the coacervate system disclosed in the present invention may be summarized as follow:
1. When components such as heme, stroma-free hemoglobin, or drugs are incorporated in the coacervate phase of the coacervate system, a true solution of a transparent, homogeneous, monomolecular dispersion results. The components are truly solubilized and cannot be filtered from the solution. Similar components added to liposomes yield a heterogeneous dispersion of liposomes in a liquid vehicle. These dispersions are not true solutions, such that liposomes, containing the added component, are suspended in a liquid vehicle and the liposomes can be separated from the liquid vehicle. PA1 2. Coacervate systems can be made from a wide variety of surface active agents, whereas true liposomes can be made only from lecithin. According to the method of the present invention, coacervate systems were prepared by using surfactants such as phospholipids, proteins, gums, synthetic polymers, polysaccarides, other natural and synthetic substances rendered surface active by chemical or physical means, and combinations thereof. If a component other than lecithin is used in an attempt to produce a liposome, an encapsulated composition, not a liposome, will result. In such instances, the encapsulating film is not a coacervate-based film. PA1 3. The preparation methods used for coacervate systems and for liposomes are different. For coacervate systems, the specific surfactant and other system components are combined such that a two-phase, liquid, aqueous system results. The physiologically active molecule is typically incorporated into the coacervate phase of the system as a process step in the production of the finished product. Liposomes, however, are made by preparing a film of lecithin and collapsing this film around the physiologically active molecule of the composition. PA1 4. When a particular component is added into the coacervate phase of a coacervate system, the exact quantity and location of the component is known. Therefore, it is possible to precisely formulate and determine the efficacy of the final product. In contrast, with liposomes, it is impossible to accurately determine the quantity and location of any added component. The precise quantity of the physiologically active molecule that adheres to the external surface or to the internal surfaces of the liposome cannot be determined. Furthermore, whenever a physiologically-active molecule having toxic potential has been incorporated in liposomes, the end-use compositions have been associated with toxicity. It has been theorized that either a toxic composition was present on the surface of the liposome or that leakage from the molecule had occurred. PA1 5. The method of manufacture of liposomes and coacervate systems are different. The preparation of a coacervate system using lecithin as a component requires relatively precise adjustment of all component concentrations and conditions, or else two-phase systems either cannot be made or can be made only with great difficulty. For liposome manufacture, precise adjustments are not necessary, because simply placing about 30% to about 50% w/v of lecithin in water will spontaneously produce a liposome. PA1 6. The stability of coacervate systems and the components added into the coacervate system is significantly greater than that of components added to liposomes. It is known that upon introduction into the body, and particularly into the circulatory system, liposomes are unstable and quickly lose their structural integrity. Therefore, because the heme molecule is also known to be very unstable, the use of an unstable liposome vehicle to incorporate unstable heme poses a variety of difficult problems.
From the foregoing facts, the composition and method of the present invention not only differs significantly from the composition described by Hasegawa, but also demonstrates a surprising and unexpected improvement over the teachings of Hasegawa.
The Kokoku Patent No. Sho. 42(1967) 117 discloses the use of a gelatin-based coacervate that incorporates hemoglobin. This Japanese reference describes particles that are approximately the size of erythrocytes (about 7 microns) and that are hardened through the use of formalin. The hardening process fixes the size and rigidity of the particles, and also results in the permanent loss of the coacervate structure. The rigidity of the particles is sufficient to prevent particle deformation that, when combined with the excessive particle size, precludes entrance of the particle into the microcirculation of the patient. In addition, it is known that particles of the size disclosed in the Japanese reference are incompatible with the reticuloendothelial system. Overall, the composition disclosed in the Japanese patent possesses an irreversible toxic characteristic. In contrast, according to the composition and method of the present invention, the coacervate structure of the composition is preserved, thereby providing microencapsulated particles that are, or are capable of distortion to, about 1 micron or less in diameter, can be prepared having substantially larger particle size to undergo distortion without loss of integrity, and are compatible with the microcirculation and the reticuloendothelial system. In addition, microencapsulated particles, of approximately 1 micron or less in diameter, are readily absorbed by the walls of the small intestine, thereby permitting oral administration of the composition. It is theorized that the microencapsulated particles of coacervated material adhere to the membrane of the gastrointestinal tract, thereby facilitating passage of the composition through the small intestine and into the circulatory system.